DESCRIPTION Intracellular switching using genetically engineered protein microdomains Biology is unparalleled in the replication of complex structures with diverse functions on the molecular and cellular scale; however, our ability to engineer functional materials at or below the size of the cell remains primitive. By using biological materials to assemble structures, process information, and harness energy, the emerging field of synthetic biology may bridge the gap between current technology and that needed to study and intervene in disease. Towards this futuristic goal, this project elaborates on a platform discovered by our team to control functional structures that are 10-1000 times smaller than a cell. These structures are based on polypeptides; therefore, they can be encoded in DNA and grown inside of living cells. Our group recently reported that temperature-responsive protein polymers expressed in the cytosol assemble organelle-sized structures within minutes of an increase of 1 degree Celsius. We named these structures genetically engineered protein microdomains, reported that they can either sort or co-assemble intact fusion proteins, and that their assembly can control a model cellular internalization pathway called clathrin-mediated endocytosis. Now we present preliminary evidence that these microdomains can activate a model cell-surface receptor and drive its internalization. This research project is designed to validate and expand the potential applications for these microdomains. The overall hypothesis is that through design, these microdomains can stimulate, deactivate, or respond to target cellular processes. Three aims are proposed: Aim 1) Manipulation of endocytotic pathways using microdomains; Aim 2) Interrogating cell signaling using ELP microdomains; and Aim 3) Expanding microdomain technology. This application innovates in three main ways: i) our interdisciplinary team is the first to report that intracellular ELPs generate microdomains that exert control over cellular pathways; ii) unlike traditional mechanisms for modulating protein activity, ELP microdomains can be activated or deactivated rapidly in live cells; and iii) this project will generalize these strategies so that they can be used to target a broad array of cellular functions. The successful demonstration of this approach is intended to shift the paradigm for how cellular biology studies are performed, enabling precise manipulation of biological processes that are fundamentally important to drug discovery. A comprehensive series of studies will be performed to demonstrate the breadth of potential applications for microdomain assembly within the cell. When completed, this project will deliver a biomolecular toolbox of broad utility to study biological processes associated with human disease.